L-tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine (serotonin), and 5-hydroxyindole acetic acid from discrete brain regions can be resolved by isocratic elution on a C-18 reverse-phase column and quantified by native fluorescence measurement. This method is rapid and simple and can reproduceably detect serotonin, tryptophan and 5-hydroxyindole acetic acid with picogram sensitivity. The detection of 5-hydroxytryptophan in brain extracts from untreated animals is difficult because of the extremely low endogenous levels of this substance in tissue. Brain tissue, urine, blood, and cerebrospinalfluid require minimal treatment for assay and the hydroxyindoles are stable enough to allow automated processing.